Surface Enhanced Raman Spectroscopy (SERS) and Surface Enhanced Resonance Raman Spectroscopy (SERRS) have been used as analytical tools for many years (1, 2). SERS active particles have been successfully employed as labels or probes in chemical assays (3), immunoassays (4, 5, 6), and DNA detection (7, 8), with the SERS peak intensity being correlated to the concentration of target species.
SERS was first discovered in 1974 by Fleischmann et al. (9) when they recorded intense Raman signals from pyridine adsorbed on roughened silver electrodes in aqueous solutions. Later, several publications concluded that these intense signals could not be accounted for by conventional Raman theory and suggested an enhancement mechanism was taking place at the metal surface, increasing the scattering intensity from each adsorbed pyridine molecule (10, 11). Since then, other publications have documented this surface enhancement effect and several theories have been proposed to account for it. Review articles describe this historical and theoretical development in detail (12, 13, 14).
Surface Enhanced Resonance Raman Spectroscopy (SERRS) combines SERS with the resonance enhancement effect associated with dye molecules (15, 16). In SERRS experiments, the dye adsorption overlaps with the Surface Plasmon Resonance (SPR) of the metal nanoparticle (NP) and the excitation laser used. The combined effect of the resonance and surface enhancement makes SERRS one of the most sensitive forms of spectroscopy available.
Both SERS and SERRS have been used for single molecule detection (17, 18). The large enhancement factors observed in SERRS experiments have been attributed to two factors, chemical enhancement and electromagnetic enhancement. Chemical enhancement is believed to result from charge transfer from the molecule to the metal surface or visa versa (19). Electromagnetic enhancement is believed to result from the localized SPR associated with the metal NP. This SPR effect greatly enhances the electromagnetic field at the surface of the NP (20). Other factors may effectively increase the Raman cross-section, such as resonance effects from a chromophore, or the presence of specific cations or anions (21).
Other publications have shown the SERS enhancement effect to be extremely large, of the order of 1012 fold, and that the use of SERRS may lead to single molecule sensitivity (22).
Unlike a fluorescent probe, a Raman probe has several structural components, such as a central nanoparticle core, a linker group, and a chromophore. Variations in the nanoparticle composition and size, the linker composition and length, and the chromophore type and number density, all potentially effect the Raman signal from the probe.
The metallic nanostructure gives rise to surface plasmon resonances. The chromophore provides chemical enhancement of the Raman signal. The energy flow and resonance coupling between the nanoparticle and the chromophore give rise to the SERRS effect. If the chromophore is too far away from the nanostructure, no coupling will occur and the Raman signal will be reduced. Chemical bonding between the chromophore and the nanoparticle also plays an important role (23).
The demonstration of the ability of SERRS to deliver single molecule sensitivity has led to much research in this area. For instance, the following US patent publications and patents provide examples of the application of this technology: U.S. Patent Application Publication No. 2007/0155021, U.S. Patent Application Publication No. 2005/0221510, U.S. Pat. No. 5,445,972, and U.S. Pat. No. 7,192,778. These SERRS applications and patents are largely directed to the use of silica surfaces coated with a metallic layer, and variations in the operation and configuration of Raman devices. Most are directed to the field of SERS active particles, as opposed to surfaces, as disclosed in U.S. Patent Application Publication No. 2007/0155021. These disclosures are based on a SERS active particle having a cationic coating. They claim that previously published methods make particles with an anionic character and that the changing of this coating to be cationic provides improvements in both signal strength and reproducibility. These prior disclosures make use of ‘surface seeking groups’ as a way of improving the Raman signal.
U.S. Pat. No. 5,445,972 discloses the use of Raman active labels attached to specific binding members for use in ligand-binding assays. However, this patent does not specify the use of metallic nanoparticles as a component of the Raman label and appears to cover the use of Raman active dye molecules, but not Raman active particles. Such techniques are a modification of the Raman dye with standard Ag surfaces or Ag nanoparticles used to obtain the full SERS signal. In these earlier techniques, Ag nanoparticles and Ag coated surfaces are used to produce the Raman signal from the Raman active label.
U.S. Pat. Nos. 7,192,778 and 6,861,263 disclose the use of metallic nanoparticles coated with a SiO2 layer. This technique comprises trapping the Raman active molecules in the SiO2 layer. The glass layer serves several functions. For instance, the glass layer keeps the Raman active molecules trapped on the surface of the nanoparticle, keeps the nanoparticle solution stable in a variety of different solvents, and acts as a point of modification when attaching other species to the whole particle structure. These patents also disclose the use of ‘sandwich’ structures, wherein the Raman active molecules are located at the junction between two metallic nanoparticles.
U.S. Patent Application Publication No. 2005/0130163 discloses a similar technique but specifies that the Raman active particle consists of more than one metallic nanoparticle dispersed in a polymer shell, wherein the polymer material may be any common polymer and glass structure.
U.S. Patent Application Publication No. 2006/0246460 describes a system for DNA detection using two Raman active molecules. In the initial state both Raman molecules are on the nanoparticle surface and so both contribute to the overall Raman signal. Upon binding the target DNA, one of the Raman molecules is removed from the nanoparticle surface and the overall Raman signal changes.
U.S. Pat. No. 6,219,137 discloses core-shell structures, but specify that the analyte provides the Raman signal. That is, the nanoparticle core shell structure has no Raman signal itself. The nanoparticle core shell structure only has a Raman signal when the target analyte binds to the metallic SERS surface. Only then is the Raman spectra obtained.